Optimization and troubleshooting in PCR.

The use of PCR to generate large amounts of a desired product can be a double-edged sword. Failure to amplify a sample under optimum conditions can lead to the generation of multiple undefined and unwanted products-even to the exclusion of the desired product. At the other extreme, no product may be amplified. A typical response at this point is to vary one or more of the many parameters that are known to contribute to primer-template fidelity and primer extension. High on the list of optimization variables are Mg 2+ concentrations, buffer pH, and cycling conditions. With regard to the last, the annealing temperature is most important. The situation is complicated further by the fact that some of the variables are quite interdependent. For example, because dNTPs directly chelate a proportional number of Mg 2+ ions, an increase in the concentration of dNTPs decreases the concentration of free Mg 2+ available to influence polymerase function.